The Paleozoic Mazon Creek Lagerstätte is one of the richest fossil assemblages and many thousands of specimens preserved in sideritic concretions have been recovered from the Francis Creek Shale of the Carbondale Formation in the Illinois Basin, United States, which is of Moscovian (∼307–311 Ma), Middle Pennsylvanian age. The concretions preserve impressions, casts, and molds of a wide variety of organisms from a broad spectrum of estuarine marine to freshwater environments (Johnson and Richardson, 1966; Nitecki, 1979; Baird et al., 1985; 1986; Clements et al., 2019). The Mazon Creek biota includes many terrestrial animals and plant remains (e.g., Wittry, 2006; 2020) that were evidently washed into coastal and marine depocenters suitable for the formation of siderite concretions in the Mazonian delta (e.g., Cotroneo et al., 2016; McCoy et al., 2016; Clements et al., 2019). Fossils in the concretions exhibit exquisite preservation which reflects the early formation of the nodules prior to significant sediment compaction and decomposition. Fossil metazoans often have the remains of soft tissues preserved. Fossil plants often contain spores (e.g., Drinnan and Crane, 1994) and also have excellent details of external morphology, which is sometimes complemented by the preservation of anatomical details (Drinnan et al., 1990). Among the many metazoans from the deposit are fossil jellyfish, polychaete worms, and the “Tully Monster” (Tullimonstrum gregarium) (Johnson and Richardson, 1966; Richardson, 1966). The latter has been studied especially intensively and the quality of preservation provides information on the probable notochord and other structures, as well as eye pigmentation, which has engendered vigorous debate on the likely systematic affinities of this enigmatic organism (e.g., Clements et al., 2016; McCoy et al., 2016; 2020; Sallan et al., 2017).

Like the fossil invertebrates and vertebrates, the floral component of the Mazon Creek biota is also diverse (Darrah, 1969; Drinnan and Crane, 1994). It includes plant parts of lycopsids, sphenopsids, ferns, pteridosperms, cordaites, as well as fossils of uncertain relationship. The fossil assemblage is dominated by leaves of ferns, sphenopsids, and pteridosperms (Locatelli et al., 2016), with lycopsids as secondary contributors (Wittry, 2006; 2020). Fossil plants in the Mazon Creek sideritic concretions exhibit excellent 3D morphology but most studies of plants from these nodules have focused on their appearance in two-dimensions and what can be seen on the surfaces of casts and molds. The first plant fossil from the Mazon Creek flora to be reconstructed in 3D was Stephanospermum konopeonus Langford, a medullosan pteridosperm ovule (Drinnan et al., 1990). Using a combination of longitudinal splitting, transverse cutting, serial sectioning and peels, Drinnan et al. (1990) characterized the histology of the ovule and reconstructed its three-dimensional form. Advanced imaging techniques including μCT now provide the opportunity to conduct similar studies non-destructively and more efficiently (Sutton, 2008), but despite the recent widespread use of μCT for reconstructing the anatomy and systematics of Mesozoic and Cenozoic plants (e.g., Friis et al., 2007; 2014; Smith et al., 2009a; Smith et al., 2009b; Collinson et al., 2013; 2016; Manchester et al., 2019; 2022; Matsunaga et al., 2019; 2021; Shi et al., 2021; Smith et al., 2021), this state-of-the-art technology has been barely applied to Mazon Creek plant fossils. To our knowledge, the only Mazon Creek plant that has been studied using a combination of light microscopy and μCT scanning is the medullosan pteridosperm ovule Stephanospermum braidwoodensis Spencer et Hilton (Spencer et al., 2013), which is very similar to Stephanospermum konopeonus.

Here, we apply these, and other advanced visualization techniques to exceptionally well-preserved fossils of Tetraphyllostrobus Gao et Zodrow and Crossotheca Zeiller from the Mazon Creek flora to determine their structure and elucidate general principles for the study of Mazon Creek fossil plants using μCT scanning.

The Mazon Creek fossil specimens studied here are housed in the paleobotanical collections of the Field Museum (FM). Three specimens recently identified as cf. Tetraphyllostrobus broganensis in Mazon Creek siderite nodules (Wittry, 2020) were examined in this study. Two (PP54628, PP58471) were scanned using a GE dual tube μCT scanner at the PaleoCT lab in the Department of Organismal Biology and Anatomy at the University of Chicago. The third (PP34514) was scanned using a Nikon XT H 225ST industrial μCT system with a Perkin Elmer 1620 X-ray detector panel and a tungsten reflection target at the University of Michigan, CTEES facility.

For PP54628 (Figure 1) both part and counterpart were scanned together separated by a thin piece of wrapping foam. The scan used a 240 kV micro-focus tube at 150kV and 250  μA at 500 m exposure timing, using filter of 0.5 mm Cu. Resolution of voxel x=y=z= 31.8520 μm with 1,200 projection images was achieved. For PP5847 (Figure 2) both part and counterpart were scanned as for PP54628, but a with 180 kV nano-focus tube at 150kV and 250  μA at 500 m exposure timing, using filter of 0.5 mm Cu. Resolution of voxel x=y=z= 20.8280 μm with 1600 projection images was achieved. PP34514 (Figure 3) was physically sectioned with a Hi-Tech Diamond 6″trim saw. Sections were scanned at 113kV and 105  μA at 500 m exposure timing, using filter of 0.5 mm Cu. Resolution of voxel x=y=z= 12.0 μm with 3,141 projection images was achieved. Anatomical details were obtained from acetate peels (Galtier et al., 1999) taken from the surfaces of individual sections and mounted on slides with Eukitt. Light photographs and microphotographs of acetate peels were obtained at the Field Museum. Macrophotography was with a digital Canon EOS R digital camera (MP-E 65 mm f/2.8 1–5x macro lens) attached to a Stackshot system, with the digital photographs merged using Helicon Focus software. Microphotography was with a ZEISS Axio Imager.A2 microscope using differential interference contrast (DIC) and a Jenoptik Gryphax NAOS digital camera.

The part and counterpart of Crossotheca (PP58059; Figure 4) were scanned together as for PP54628 and PP58471, but with a 180 kV nano-focus tube, at 150 kV and 250 μA at 500 m exposure timing, using filter of 0.2 mm Cu. Resolution of voxel x=y=z= 32.2230 μm with 1,800 projection images was achieved.

The μCT volumes were visualized mainly using Avizo Software 3D 2022.1 (Thermo Fisher Scientific, Waltham, MA, United States). A volume thresholding tool in Avizo was used to demarcate the digitized 3D volume of the fossil specimens from the surrounding matrix of the concretion. Multiple functions were applied to the data to briefly evaluate the quality of the scans and the 3D preservation using volrens, isosurfaces, isosurface renderings, and volume renderings. An isosurface was first created to reconstruct the buried surface of the parts and counterparts (draw style: shaded) with the “back face” function selected. Once the desired surface models were achieved, each volume was exported as a .ply file for further analysis or visualization in MeshLab 2022.02 (Cignoni et al., 2008). Additional filters in MeshLab were helpful for removal of isolated surface fragments. MeshLab rendering-shaders functions, for example, depthmap, slicingplane, radiancescaling (lit-sphere effect), and x-ray were applied to improve the visualization of the surface models.

In-situ spores from the permineralized cone of specimen PP34514 (Figure 3) were examined directly on polished specimens attached to a stub and scanned at the National Museum of Natural History with a Zeiss EVO MA15 scanning electron microscope (EHT = 12 kv, WD = 7.5 mm, I probe = 1.2 nA, signalA = CZBSD). The spores were also imaged using a Zeiss LSM 980 with Airyscan 2 confocal superresolution microscope at the Smithsonian Tropical Research Institute. Images were taken at ×630 magnification (×63/NA 1.4 oil DIC) with fluorescence contrast, using 561 and 639 nm laser wavelengths and an intensity of 40% and 16% respectively. A total of 147 axial images were taken at 0.19 μm increments to capture the 3D morphology of each spore. Images were analyzed using Zeiss Zen 3.4.

To evaluate the response to μCT of specimens preserved in different minerals we acquired in situ and ex-situ elemental maps (Oxford Instruments AZTec) of the fossil concretions (Supplementary Material). In-situ and ex-situ Raman microspectroscopy was performed at FM with a WITec alpha 300 R system equipped with a 532 nm laser (Supplementary Material). Data was analyzed using WITec Project Plus software.

Biomarkers and macromolecular biosignatures obtained with in situ Raman spectra studies on vertebrates, invertebrates, and coprolites from Mazon Creek have recently provided exciting results for reevaluating the affinity of problematic fossil organisms (McCoy et al., 2020) and the reconstruction of trophic relationships in this Carboniferous ecosystem (Tripp et al., 2022). Raman analyses showed the potential utility of this technique for recognizing the mineralogical composition of Mazon Creek fossil plants, but future biomarker analyses could also help elucidate the deep relationships among vascular plants and other unexplored trophic plant dynamic processes.

Approximately eighty plant taxa have been recognized from the Mazon Creek Lagerstätte, and there are more than 20,000 plant fossil specimens in the Field Museum collections. However, many of the plant taxa require careful structural analysis and systematic revaluation, including the specimens of Tetraphyllostrobus and Crossotheca studied here. A previous study by Drinnan et al. (1990) on ovules of Stephanospermum konopeonus demonstrated the potential for extracting high quality 3D information from plant fossils preserved in Mazon Creek ovules. More recently a study by Spencer et al. (2013) on a similar medullosan ovule, Stephanospermum braidwoodensis, showed that non-destructive μCT further enhanced the opportunities to develop 3D reconstructions of fossil plants preserved in siderite nodules. The current study on other kinds of plant fossils in Mazon Creek nodules further demonstrates the value of μCT scanning and the resulting visualizations for understanding the 3D structure of Paleozoic plants. However, the quality of results will be influenced by the composition of the minerals filling the voids left by the decomposing plant material, with calcite, aluminosilicates, iron oxides, sulfides, and pyrite all potentially involved. For 3D renderings of Mazon Creek plants, specimens with high amounts of organic preservation and more homogeneous mineralization should be prioritized, with sulfides preferred over iron oxides. Specimens in which the voids are filled in part by aluminosilicates are likely to give the least helpful results. Excellent preservation of spores studied with DIC, SEM, and Airyscan confocal super-resolution microscopy also provides useful high-quality information for the correct identification of in situ palynomorphs.